Zhang Hongming, Raji Abayami, Theisen Michael, Hansen Paul R, Marconi Richard T
Department of Microbiology and Immunology, Center for the Study of Biological Complexity, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.
J Bacteriol. 2005 Jan;187(1):175-84. doi: 10.1128/JB.187.1.175-184.2005.
The Bdr proteins are polymorphic inner membrane proteins produced by most Borrelia species. In Borrelia burgdorferi B31MI, the18 bdr genes form three subfamilies, bdrD, bdrE, and bdrF. The production of at least one of the Bdr paralogs, BdrF2, is up-regulated in host-adapted spirochetes, suggesting a role for the protein in the mammalian environment. Here, we demonstrate using reverse transcriptase (RT) PCR that BBG29, BBG30, BBG31, and BBG32, which reside upstream of bdrF2, are cotranscribed with bdrF2 as a five-gene operon. While the functions of most of these proteins are unknown, BBG32 encodes a putative DNA helicase. Real-time RT-PCR analyses demonstrated higher levels of bdrF2 transcript relative to other genes of the operon, suggesting that bdrF2 may also be transcribed independently from an internal promoter. Internal promoters were detected using the 5' rapid amplification of cDNA ends system. The putative promoter associated with bdrF2 was found to be highly similar in sequence to the multiple promoters associated with the ospC gene. Real-time RT-PCR analyses, performed to assess the expression of these genes in infected mice, revealed that genes of the bdrF2 locus are expressed only during early infection, suggesting a role in the establishment of infection. To further characterize the proteins encoded by the bdrF2 locus, which have unknown functions, the cellular localizations of these proteins were determined by Triton X-114 extraction and phase partitioning. BBG29 and BBG31 were found to be cytoplasmic. To determine if these proteins elicit an antibody (Ab) response during infection, immunoblot analyses were performed. Abs to these proteins were not detected. Based on the analyses presented here, we offer the hypothesis that BdrF2 and other proteins encoded by the operon form an inner-membrane-associated protein complex that may interact with DNA and which carries out its functional role during transmission or the early stages of infection.
Bdr蛋白是大多数疏螺旋体物种产生的多态性内膜蛋白。在伯氏疏螺旋体B31MI中，18个bdr基因形成三个亚家族，即bdrD、bdrE和bdrF。至少一种Bdr旁系同源物BdrF2在适应宿主的螺旋体中表达上调，这表明该蛋白在哺乳动物环境中发挥作用。在这里，我们使用逆转录酶（RT）PCR证明，位于bdrF2上游的BBG29、BBG30、BBG31和BBG32与bdrF2一起作为一个五基因操纵子进行共转录。虽然这些蛋白中的大多数功能未知，但BBG32编码一种假定的DNA解旋酶。实时RT-PCR分析表明，相对于操纵子的其他基因，bdrF2转录本水平更高,这表明bdrF2也可能从内部启动子独立转录。使用5' cDNA末端快速扩增系统检测到内部启动子。发现与bdrF2相关的假定启动子在序列上与与ospC基因相关的多个启动子高度相似。为评估这些基因在感染小鼠中的表达而进行的实时RT-PCR分析表明，bdrF2基因座的基因仅在早期感染期间表达，这表明其在感染建立过程中发挥作用。为了进一步表征bdrF2基因座编码的功能未知的蛋白质，通过Triton X-114提取和相分配确定了这些蛋白质的细胞定位。发现BBG29和BBG31位于细胞质中。为了确定这些蛋白在感染期间是否引发抗体（Ab）反应，进行了免疫印迹分析。未检测到针对这些蛋白的抗体。基于此处给出的分析，我们提出假说，即BdrF2和操纵子编码的其他蛋白形成一种与内膜相关的蛋白复合物，该复合物可能与DNA相互作用，并在传播或感染早期发挥其功能作用。
